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Schistosoma japonicum katsurada recombinant protein SjSAPLP5 as well as encoding gene and application thereof

A recombinant protein, schistosomiasis technology, applied in recombinant DNA technology, application, genetic engineering and other directions, to achieve the effect of good immunogenicity and good antigenicity

Active Publication Date: 2016-01-20
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the sensitivity (90%) of the diagnostic kit still has room for further improvement

Method used

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  • Schistosoma japonicum katsurada recombinant protein SjSAPLP5 as well as encoding gene and application thereof
  • Schistosoma japonicum katsurada recombinant protein SjSAPLP5 as well as encoding gene and application thereof
  • Schistosoma japonicum katsurada recombinant protein SjSAPLP5 as well as encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Cloning of SjSAPLP5 gene of Schistosoma japonicum

[0046] According to the sequence of SjSAPLP5 gene (GenBankFN315317.1) (as shown in SEQIDNO.1), design primers and introduce restriction sites, as follows:

[0047] PF: 5’-CGC GGATCC TCAACATTGAACAGTAATAAT-3' (as shown in SEQ ID NO.3, the italicized part represents the protective base of the upstream primer, and the underlined part is the BamHI restriction site of the upstream primer);

[0048] PR: 5’-CTG CTCGAG TGCAAAAGGATATTTCAAA-3' (as shown in SEQ ID NO.4, the italicized part represents the protective base of the downstream primer, and the underlined part is the XhoI restriction site of the downstream primer);

[0049] The specific primers were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0050] Using the cDNA of 42-day male and female adults of Schistosoma japonicum as a template, PCR was performed to amplify the ORF fragment of SjSAPLP5 gene. The reaction system was as follows: high-fidelity DNA polym...

Embodiment 2

[0054] Example 2 Expression and purification of SjSAPLP5 recombinant protein of Schistosoma japonicum

[0055] Transform the correctly sequenced pET28a(+)-SjSAPLP5 recombinant plasmid to express competent cells Transetta(DE3) (Beijing Quanshijin Biotechnology Co., Ltd.); inoculate the positive clones by PCR into LB liquid medium (containing 50μg) / ml kanamycin) 15mL, incubate overnight at 37°C, transfer 10mL medium to 1L of LB medium (containing 50μg / ml kanamycin) the next day, and continue to cultivate to OD 600nm The value was 0.8, and IPTG with a final concentration of 1 mM was added to induce expression for 4 hours, and the cells were collected by centrifugation, and frozen at -80°C for use.

[0056] Suspend a small amount of bacteria before and after induction in PBS buffer, add SDS-PAGE loading buffer, mix well and cook for 5 minutes in a boiling water bath to denature the protein. Add 10 μl of the sample before and after induction to each sample well, and perform SDS-PAGE a...

Embodiment 3

[0058] Example 3 Antigenicity detection of SjSAPLP5 recombinant protein of Schistosoma japonicum

[0059] SDS-PAGE electrophoresis: 100ng of the SjSAPLP5 recombinant protein obtained in Example 2 was loaded and the electrophoresis conditions were: 100V20min, 120V1h.

[0060] Transfer membrane: Use the wet transfer method to transfer the protein in the PAGE gel to the PVDF membrane. The electrotransfer conditions are: ice bath, 100V1h.

[0061] Blocking: Block the PVDF membrane with 5% skimmed milk powder at room temperature for 2 hours, and wash 3 times with TBST buffer.

[0062] Incubation with primary antibody: add the serum of BALB / c mice infected with Schistosoma japonicum for 42 days, New Zealand white rabbit serum from Schistosoma japonicum for 42 days, and the serum of Schistosoma japonicum patients, with mouse anti-His-Tag antibody (Abimart Biomed Company) as a positive control, healthy mouse serum as a negative control (diluted with blocking solution 1:500), incubated overnig...

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Abstract

The invention provides a schistosoma japonicum katsurada recombinant protein SjSAPLP5 as well as an encoding gene and an application thereof. The protein provided by the invention has amino acid sequence as shown in SEQ ID No.2 or has amino acid sequence which is formed by replacement, deletion and / or addition of one or more of amino acid residues and has the same function. The invention also provides the encoding gene for encoding the protein. The recombinant protein SjSAPLP5 provided by the invention is excellent in immunogenicity, can be served as an excellent diagnosis antigen, is used for preparing a schistosoma japonicum katsurada diagnostic kit with high sensitivity and high specificity, and is also used for preparing an anti-schistosome vaccine and the drug served as a potential drug effecting target spot and is used for screening and treating the schistosoma japonicum katsurada diseases.

Description

Technical field [0001] The invention relates to the field of biotechnology, in particular to a Schistosoma japonicum recombinant protein SjSAPLP5 and its coding gene and application. Background technique [0002] Schistosomiasis is an infectious parasitic disease that seriously threatens human health. It is mainly prevalent in 76 countries and regions in Asia, Africa and Latin America. More than 200 million people are infected worldwide, and nearly 800 million people are threatened by its infection. Schistosomiasis japonicum is prevalent in my country. After decades of unremitting efforts, my country's schistosomiasis prevention and control work has achieved world-renowned achievements. Schistosomiasis has been controlled in endemic areas, but there is still a heavy task to achieve the ultimate goal of eliminating schistosomiasis far. [0003] Diagnosis is the central link in the field of schistosomiasis control. Accurate diagnostic technology not only has important clinical signi...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/12C12N15/11G01N33/68A61K39/00A61P33/12
CPCA61K39/00C07K14/43559G01N33/68G01N2333/43547Y02A50/30
Inventor 陈启军刘帅
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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